One of the main challenges facing the tomato industry is how to deliver to a processing plant or to the marketplace tomato fruit that have been vine-ripened (i.e., desirable to consumers in taste, texture, and color) but that remain firm without the usual softening that reduces the shelf life of harvested fruit. Because traditional breeding methods are very labor intensive, it could take years to develop a novel tomato variety that may display only a modest increase in shelf life. Recent studies have utilized genetic and biochemical techniques in an effort to identify the factors that affect fruit softening. By identifying and modifying the expression of specific genes that are involved in cell wall degradation, researchers and breeders hope to develop new tomato varieties that have the desirable qualities of vine-ripened fruit, but that also are resistant to post-harvest softening and, therefore, display a longer shelf life with reduced spoilage.
Fruit softening is one of the many ripening-related changes, including alterations in fruit texture, color, aroma, and metabolism of sugars and organic acids, which occur as a result of a developmental program triggered by ethylene. Recent data indicate that cell wall proteins called expansins are important regulators of fruit softening in tomato fruit. This large multigene family of proteins has been proposed to loosen cell walls and stimulate plant cell enlargement by weakening the non-covalent bonds between glucans. The observation that mRNA and protein for LeExp1, the primary expansin expressed in tomato fruit, is upregulated with ripening led to the hypothesis that this protein is involved in cell wall disassembly. Consistent with this idea, treatment of green wild type fruit with ethylene gas results in a rapid and robust rise in LeExp1 mRNA whereas LeExp1 expression levels are not increased by ethylene in ripening-impaired rin mutant tomatoes (Rose et al., Proceedings of the National Academy of Sciences USA 94:5955-5960, 1997).
Antisense expression of a LeExp1 transgene in tomato plants has confirmed the importance of the expansins to the commercial tomato industry. Fruit of tomato plants expressing an antisense LeExp1 transgene under the direction of a constitutively expressed promoter have reduced endogenous Exp1 levels and increased firmness compared to wild type tomato fruit. In contrast, expression of a sense LeExp1 transgene increased Exp1 mRNA and protein levels in tomato fruit and enhanced fruit softening (Brummell et al., The Plant Cell 11:2203-2216, 1999; U.S. Pat. No. 6,350,935). An antisense LeExp1 transgene also affects tomato processing qualities for juice and paste (Kalamaki et al., Journal of Agricultural and Food Chemistry 51(25):7465-7471, 2003; Kalamaki et al., Journal of Agricultural and Food Chemistry 51(25):7456-7464, 2003).
These data suggest that modulation of LeExp1 levels in tomatoes affects fruit softening, a key factor that limits the shelf life of fresh tomatoes. However, numerous expansins with overlapping patterns of expression are detectable in tomato fruit during development. This observation opens the possibility that the antisense LeExp1 transgene reduces not only LeExp1, but also suppresses the expression of other expansins. The method described herein specifically targets the LeExp1 gene and plants generated by this method contain mutations in LeExp1.
Transgenic technology has successfully utilized antisense LeExp1 transgenes to reduce post-harvest softening in tomato fruit. However, public acceptance of genetically modified plants, particularly with respect to plants used for food, is not universal. Alternatively, traditional breeding methods could be used to develop new tomato varieties with reduced expansin protein levels or activity. However, these methods are both laborious and time-consuming. In addition, undesirable characteristics often are transferred along with the desired traits when tomato plants are crossed in traditional breeding programs.
Because some consumers have clear preferences against genetically modified foods, it would be useful to have a tomato that exhibits reduced levels of LeExp1, but that is not the result of genetic engineering. However, to date, a naturally occurring “knockout” or “knockdown” of any endogenous tomato expansin gene is not known in the art. The inventors have screened an 802 base pair region of the LeExp1 gene in 183 commercial, heirloom and collected tomato varieties to assess existing natural genetic variation. The inventors uncovered one mutation in an intronic region of the LeExp1 gene, but no mutations in its coding region. These findings indicate the lack of natural genetic variation in the LeExp1 gene of germplasm that is available to tomato breeders. The availability of multiple allelic mutations in LeExp1 would provide tomato breeders with novel genetic variation and a spectrum of phenotypes for the development of new firmer fleshed tomato varieties. A cultivated tomato with reduced fruit softening as a result of its LeExp1 gene either knocked out or otherwise hindered that was not the result of genetic engineering would have tremendous value for the tomato industry, including fresh market tomatoes, processor tomatoes and tomato food products such as sliced tomatoes, canned tomatoes, ketchups, soups, sauces, juices and pastes.